Field of the Invention
This invention relates genetically altered alfalfa plants that contain heterologous cDNA encoding hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase (HDT) and produce clovamide and/or related hydroxycinnamoyl amides. This invention also relates to the cDNAs that encode HDT1 and HDT2 obtained from red clover, expression vectors containing the cDNAs, and the use of the cDNAs and/or expression vectors to genetically modify alfalfa so that the modified alfalfa can produce clovamide and/or related hydroxycinnamoyl amides. This invention also relates to genetically altered alfalfa that have a phenotype involving post-harvest protein protection.
Description of Related Art
Clovamide (an amide formed between caffeic acid and L-DOPA [L-3,4-dihydroxyphenylalanine]) is one of two major o-diphenol compounds present in leaves of red clover. The other major o-diphenol compound present in red clover leaves is phaselic acid (an ester formed between caffeic acid and malic acid). When oxidized by the endogenous polyphenol oxidase system (PPO), caffeic acid derivatives such as phaselic acid and clovamide constitute a natural system of post-harvest protein protection for forage crops. See U.S. Pat. No. 8,338,339.
Unfortunately, many important forages, like alfalfa, do not make PPO or the o-diphenol compounds needed for this process. For alfalfa alone, post-harvest proteolytic losses upon harvest and storage as silage cost U.S. farmers an estimated $100 million annually. Poor utilization of degraded forage protein by ruminant animals also results in release of the excess nitrogen into the environment as urea. Forages rich in PPO and o-diphenols appear to have reduced protein and lipid degradation in the rumen, with the potential for additional nitrogen utilization efficiency and improved lipid profiles of animal products, respectively (see, Lee et al., 2004, J. Sci. Food Agric. 84:1639).
Mature red clover leaves accumulate relatively high levels of two caffeic acid derivatives: phaselic acid (an ester formed between caffeic acid and malic acid) (5 to 8 mmol/kg fresh weight [FW]), and clovamide (an amide formed between caffeic acid and the amino acid L-DOPA) (3 to 6 mmol/kg FW) (Sullivan and Zeller, 2013, J. Sci. Food. Agri. 93(2):219-26). Previously, a red clover gene (HCT2, Genbank EU861219) encoding a hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase (HMT) was shown to be crucial for phaselic acid accumulation in red clover leaves (Sullivan and Zarnowski, 2011, Plant Physiol. 155(3):1060-7).
Although in red clover, phaselic acid is a major hydroxycinnamoyl-malate ester, expression of red clover HCT2 in alfalfa results in mostly accumulation of p-coumaroyl-malate and feruloyl-malate, compounds that do not function with PPO to preserve forage protein. See, Sullivan, M., (2015) “Engineering alfalfa to accumulate useful caffeic acid derivatives and characterization of hydroxycinnamoyl-CoA transferases from legumes” in The Phytochemical Society of North America, Aug. 8-12, 2015 (conferences.illinois.edu/psna/documents/PSNA_2015_Full_Program.pdf). This accumulation pattern in alfalfa is actually consistent with the in-vitro enzymatic properties of HCT2 gene product (HMT) whereby p-coumaroyl-CoA and feruloyl-CoA donor substrates are preferred over caffeoyl-CoA by five- to tenfold (see Sullivan and Zarnowski, 2011).
Thus, a need exists for genetically altered alfalfa plants that can produce clovamide and/or related hydroxycinnamoyl amides which can protect proteins from post-harvest degradation. Such a genetically altered alfalfa plant must contain cDNA encoding the appropriate enzyme which produces clovamide. Based on the research presented below, that enzyme is termed hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl transferase (HDT).